Project Summary Fanconi anemia (FA) is a recessive disorder caused by deficient DNA damage repair. FA patients exhibit aplastic anemia, congenital abnormalities, and profoundly elevated cancer occurrence. Cells derived from FA patients are hypersensitivity to DNA crosslinking agents and highly susceptible to chromosome breakage under genotoxic stress. To date, 15 autosomal and 1 X-linked genes are designated as causative genes for FA, but the molecular structure of the FA pathway remains largely unclear. Lack of defined genetic model systems and a scarcity of recognizable protein domains in most FA proteins are among the major obstacles impeding the advance of FA biology. The main objective of this proposal is to establish the genetic framework of the FA pathway and to elucidate molecular functions of key FA proteins. We begin to approach these problems by systematically constructing somatic cellular knockout models. Our preliminary investigations insinuate a hypothesis that different FA proteins form distinct functional modules to accomplish the DNA damage-induced FA pathway activation, which enables the recruitment of DNA damage-processing enzymes. We plan to test this hypothesis with three specific aims: (1) Define the epistatic relationships among classic FA gene products, which will mitigate a visible void in genetic connections among Fanconi anemia genes. (2) Dissect the functional integration of the FA core E3 ligase complex which contains several FA proteins with unknown functions. (3) Determine whether DNA-protein crosslinks are prevalent endogenous lesions processed by the FA pathway. Elucidation of the FA pathway should have a significant impact in advancing the understanding of fundamental cellular mechanisms protecting genome integrity. More importantly, FA pathway components are potential target for therapeutic intervention. The FA pathway functions primarily in resolving replication fork- blocking DNA lesions. This type of lesions is exemplified by DNA crosslinks most frequently generated by bifunctional alkylating chemotherapeutic modalities, such cisplatin and melphalan, and by DNA-protein crosslinks produced with high frequency from ionizing radiation exposure. For example, clinical response of many ovarian cancers to cisplatin treatment is dictated by their FA pathway status. In summary, this project is aimed at delineating the molecular pathological mechanism of Fanconi anemia with the immediate benefit of uncovering novel therapeutic targets to the improve cancer treatment outcomes.